1. Field of the Invention
The present invention relates, in general, to preparing blood spreads for microscopical examination and, in particular, to blood spreads having a smooth and homogeneous plasma layer. More specifically, this invention relates to a process comprising a combination of steps including timely fixation for the purpose of cell preservation, producing a cross-linked matrix in the plasma and performing of chemical reactions by diffusing reagents across an interface, for the purpose of stabilizing both the cell shape and the smoothness and homogeneity of the plasma layer, while the fresh and still wet blood spread is immersed in a non-polar solvent.
2. Description of the Prior Art
In the routine examination of blood for diagnostic purposes, it has been customary to spread a thin layer over the surface of a glass slide. A portion of the spread, referred to as a monolayer, is examined microscopically after a suitable process of staining which brings out contrast. Monolayer, in this context, is a reference to there being a single layer of cells without significant overlap. In the past, the plasma layer, which separates cells in this monolayer, has been acknowledged, but not regarded as having much importance either in the process of spreading the monolayer or in the subsequent analysis.
Three commonly-accepted methods of preparing blood spreads for microscopical examination are the Coverslip Method, the Wedge Smear Method and the Spinner Method. These are generally described in U.S. Pat. No. 4,209,548 as well as in U.S. Pat. Nos. 3,827,805 and 4,108,109. As described in U.S. Pat. No. 4,209,548, which teaches a method of vaporous fixation of the wet spread in the time between spreading and drying, there is a tendency to distort the shape of red blood cells during the process of drying the sample after formation of the monolayer by any means. This same patent also indicates in column 2, line 26, that cells would wash off the slide if the slide were dipped in a fixing solution before drying takes place. The process of vaporous fixation was also practiced by Adler, Saunders and Ornstein in a device described in a 1977 publication (Adler, S. L., Saunders, A., and Ornstein, L., "Fully Automated Preparation of High Quality Stained Blood Films," Advances in Automated Analysis, Mediad, Inc., Vol. I. 1977, pp. 77-80).
There are several disadvantages of preserving the shape of red blood cells by vaporous fixing of the monolayer. First, there are harmful effects on the shape of white blood cells which are not permitted to spread widely on the slide. Secondly, one is required to hold the slide in a vapor chamber for an extended amount of time. Since this chamber is the apparatus for making the monolayer, it is inoperable for a second specimen during processing of the first. Thirdly, the whole slide is necessarily exposed to the vapor and, hence, if the vapor is harmful to any part of the subsequent examination, a second slide must be prepared, excluding the vapor. Further, the vapor is toxic and therefore a health hazard unless carefully controlled.
During the performance of experiments on blood samples with artificially colored plasma, it was observed that the drying process involves variable degrees of migration of the plasma. This migration is best visualized by having the freshly prepared slide under microscopic observation while it is still wet. It is then possible to observe a timed sequence, in which the plasma layer appears smooth and homogeneous at first. Over a period of 15 seconds or less the colored plasma layer flows toward any proximal cell or particle. This migration stops when the specimen is dry. At this time it is possible to observe that the colored plasma has migrated to form rings around the cells or particles on the glass surface. Other portions of the colored plasma residue had formed irregular patterns between cells during the drying process.
By experiment it has been possible to show that changing the surface tension characteristics of the plasma provides some control over the degree of migration of colored plasma during the drying process. It was concluded that surface tension forces have a major influence during the drying process, both on the liquid plasma and on the shape of cells adherent to the slide.
Control of surface tension is possible by the addition of surface active agents prior to the application of blood to the slide. The surface active agents may be added either to the slide surface or to the blood. Such agents, however, also have a rapid and harmful effect on the surface membrane of cells and, therefore, cause a different and still unacceptable distortion of shape of both red blood cells and white blood cells.
Control of surface tension is also possible by immersing the slide while the spread sample is still wet. Since there is no surface, the forces are avoided. By immersing a still wet slide in a non-polar (non-water miscible) solvent such as cyclohexane, it has been possible to inhibit the migration of colored plasma for as much as 5 to 10 minutes. As the slide is removed from cyclohexane, however, and the solvent evaporates, the migration of colored plasma resumes and forms a ring around nearby cells or some irregular pattern. Thus, the process of distortion is retarded but not eliminated by simply immersing the wet slide in a nonmiscible (non-polar) solvent.
A process of cell embedding on the slide surface was described by L. Ornstein in "Technicon Autoslide Staining and Processing System," Advances in Automated Analysis, Mediad, Inc., Vol. I, 1977, pp. 81-82. In this process, however, the cells were already dry before infiltrating with an acrylic monomer and subsequently polymerizing with ultra violet light.
With respect to the action of a reagent across an interface, the synthesis of nylon, for example, to surround an aqueous droplet is practiced as one form of microencapsulation. For this purpose, the dispersed microdroplets of water contain the reagent hexamethylene diamine, while the non-polar solvent in which the microdroplets are dispersed may, for example, be a mixture of cyclohexane and chloroform. At the proper time during the dispersion process, the reagent sebacoyl chloride is added to the non-polar solvent. Reaction between the two active agents, hexamethylene diamine and sebacoyl chloride takes place at the interface between water and the non-polar solvent. The result is formation of a thin layer or membrane which completely surrounds the water droplets. Thickness can be controlled by concentration of the reagents and by time of reaction (See Immobilized Enzymes, CRC Press, 1973). However, when this process is performed on a wet blood film on a slide, the nylon formed by the reaction remains as a membrane at the interface. It covers but does not embed the cells and plasma and does not produce a matrix in the plasma layer.
It is, therefore, an object of this invention to provide a process of preparing blood spreads for microscopical examination which is devoid of the above-noted disadvantages.
It is another object of this invention to provide a process of preparing blood spreads wherein there is no distortion of the plasma layer during drying of plasma or of cell monolayers.
It is still another object of the present invention to provide a process of preparing blood films wherein the red blood cells and white blood cells are preserved in their original shape on the slide.
It is a further object of the present invention to provide a process of preparing blood films wherein both a smooth and undistorted plasma layer and cells without distortion are preserved.